Combined cancer therapy with immune checkpoint modulators and fermentation products by symbiotic microbiota

ABSTRACT

Combined therapy of cancer using an immune check point modulators (e.g., an immune checkpoint inhibitor) and a fermented product, which may be prepared using symbiotic microbiota.

RELATED APPLICATION

This application claims the benefit of U.S. provisional application No.62/315,259, filed Mar. 30, 2016 under 35 U.S.C. § 119, the entirecontent of which is herein incorporated by reference.

BACKGROUND OF THE INVENTION

Immune checkpoints are molecules that regulate signaling pathways in theimmune system, thereby regulating immune cell activities in immuneresponses. Many cancer cells can escape immune surveillance viainhibiting T cell activation. Since 2010, immune checkpoints have beenincreasingly considered as new targets for cancer immunotherapy. Anumber of checkpoint modulators, e.g., Yervoy® (ipilimumab), Keytruda®(pembrolizumab), and Opdivo® (nivolumab), have been demonstrated inclinical trials as being effective in suppressing cancer cell growth.

Fermentation is a metabolic process that converts sugar to acids, gasesor alcohol by microorganisms, such as yeast and bacteria, or byoxygen-starved muscle cells, as in the case of lactic acid fermentation.In food processing, fermentation is the conversion of carbohydrates toalcohols and carbon dioxide or organic acids using yeasts, bacteria, ora combination thereof, under anaerobic conditions. Metabolites generatedin a fermentation process often confer benefits to health.

SUMMARY OF THE INVENTION

The present disclosure is based on the unexpected synergistic effects ofa combination of an immune check point modulator (e.g., an anti-PD1antibody) and a fermented soybean composition on suppressing cancergrowth.

Accordingly, one aspect of the present disclosure provides a method fortreating cancer, comprising: (i) administering to a subject in needthereof an effective amount of an immune checkpoint modulator (e.g., animmune checkpoint inhibitor); and (ii) administering to the subject afermented composition (e.g., a fermented soybean composition), whichcomprises multiple metabolites that are generated via fermentation of alegume plant, a portion thereof, or an extract thereof by one or moresuitable microorganisms.

In another aspect, the present disclosure provides a method for treatingcancer, comprising administering to a subject in need thereof aneffective amount of a fermented soybean composition as described herein,wherein the subject has undergone or is undergoing an anti-cancertherapy that involves an immune checkpoint modulator (e.g., an immunecheckpoint inhibitor).

In any of the methods described herein, the fermented composition can beadministered by oral administration or by intravenous administration. Insome examples, the fermented composition can be in liquid form. In someembodiments, the fermented composition may comprise multiple metabolitesthat are generated via fermentation of a legume plant (e.g., soybean), aportion thereof (e.g., seeds), or an extract thereof by a yeast, alactobacillus, or a combination thereof. The fermented composition maybeadministered before, after, or concurrently with the administration ofthe immune checkpoint modulator.

In some embodiments, the fermented composition may comprise acombination of (e.g., two or more) of lactic acid, acetic acid, and3-aminoisobutyric acid. In one example, the fermented compositioncomprise lactic acid at 5-20% by weight, acetic acid at less than 5% byweight, and 3-aminoisobutyric acid at less than 5% by weight.

In some examples, the fermented composition may be prepared by a processcomprising: (i) growing the yeast, the lactobacillus, or the combinationthereof in a medium comprising a legume plant (e.g., soybean), a portionthereof (e.g., seeds), or an extract thereof under conditions allowingfor fermentation of the soybean or the extract thereof; and (ii)collecting the fermented composition obtained from step (i). Optionally,the preparation process may further comprise filtering the fermentedcomposition, sterilizing the fermented composition, and/or concentratingthe fermented composition.

The immune checkpoint modulator for use in any of the methods describedherein can be an modulator (e.g., an inhibitor) of an immune checkpointmolecule, which may be PD-1, CD28, CTLA-4, CD137, CD40, CD134, ICOS,KIR, LAG3, CD27, TIM-3, BTLA, GITR, TIGIT, CD96, CD226, KIR2DL, VISTA,HLLA2, TLIA, DNAM-1, CEACAM1, CD155, IDO (e.g., IDO1), TGF-beta, IL-10,IL-2, IL-15, CSF-1, IL-6, and adenosine A2A receptor (A2AR), or a ligandthereof. In some embodiments, the immune checkpoint modulator is anantibody specific to the immune checkpoint, or a ligand thereof, forexample, an antibody specific to PD1 or a ligand thereof (PDL1 or PDL2).In some examples, the antibody can be a human antibody, a humanizedantibody, or a chimeric antibody.

Exemplary cancers to be treated by the method described herein include,but are not limited to, colon cancer, lung cancer, breast cancer,pancreatic cancer, skin cancer, brain cancer, ovarian cancer, kidneycancer, stomach cancer, head and neck cancer, esophageal cancer, bladdercancer, rectal cancer, bone cancer, uterine cancer, prostate cancer, andhematological malignancy.

Further, the present disclosure features a kit comprising (i) one ormore of the immune checkpoint modulators as described herein, and (ii) afermented soybean composition as described herein.

Also within the scope of the present disclosure are (i) pharmaceuticalcompositions for use in treating cancer, e.g., those described herein,the pharmaceutical composition comprising an immune checkpoint modulatorand a fermented soybean composition as described herein; and uses of thecombination of the immune checkpoint modulator and fermented soybeancomposition for manufacturing a medicament for use in treating cancer.

The details of one or more embodiments of the invention are set forth inthe description below. Other features or advantages of the presentinvention will be apparent from the following drawings and detaileddescription of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a schematic illustration of an experimental design forstudying the effect of a combination of an anti-PD1 antibody and afermented composition (Composition X) via oral administration in a coloncancer mouse model. The mice were concurrently transplanted with coloncancer CT26 cells and treated with of the fermented composition.

FIG. 1B is a schematic illustration of an experimental design forstudying the effect of a combination of an anti-PD1 antibody and afermented composition (Composition X) via oral administration in a coloncancer mouse model. The mice were treated with the fermented compositionprior to the transplantation of colon cancer CT26 cells.

FIG. 2A is a photo showing sizes of tumors formed by subcutaneousinjection of colon cancer CT26 cells in mice treated with vehiclecontrol, an anti-PD1 antibody, or combinations of the anti-PD1 antibodyand a fermented composition as various doses. The mice were concurrentlytransplanted with colon cancer CT26 cells and treated with of thefermented composition. No significant difference in body weight wasobserved among the groups.

FIG. 2B is a photo showing sizes of tumors formed by subcutaneousinjection of colon cancer CT26 cells in mice treated with vehiclecontrol, an anti-PD1 antibody, or combinations of the anti-PD1 antibodyand a fermented composition as various doses. The mice were treated withthe fermented composition prior to the transplantation of colon cancerCT26 cells. No significant difference in body weight was observed amongthe groups.

FIG. 3A includes charts showing the inhibitory effects of theanti-PD1/Composition X combination on tumor growth observed in a coloncancer mouse model. The mice were concurrently transplanted with coloncancer CT26 cells and treated with of the fermented composition.

FIG. 3B includes charts showing the inhibitory effects of theanti-PD1/Composition X combination on tumor growth observed in a coloncancer mouse model. The mice were treated with the fermented compositionprior to the transplantation of colon cancer CT26 cells.

FIG. 4 includes charts showing the effect of Composition X-anti-PD1combinations on splenic T cell profiling observed in a colon cancermouse model. *p<0.05; **p<0.01; and ***p<0.001. P-values were obtainedby comparison of each intervention group and the control group (vehicletreated). #p<0.05; ##p<0.01; ###p<0.001. P-values were obtained bycomparison of each intervention group and the anti-PD1 antibody-treatedgroup.

FIG. 5 is a schematic illustration of an exemplary experimental designfor studying the effect of Composition X-anti-PD1 combinations in a lungcancer mouse model.

FIG. 6 is a photo showing sizes of tumors formed by subcutaneousinjection of lung carcinoma LL2 cells in mice treated with vehiclecontrol, an anti-PD1 antibody, or combinations of the anti-PD1 antibodyand a fermented composition as various doses. No significant differencein body weight was observed among the groups.

FIG. 7 includes charts showing the inhibitory effects of theanti-PD1/Composition X combination on tumor growth observed in a lungcancer mouse model.

FIG. 8 includes charts showing the effect of Composition X-anti-PD1combinations on tumor-infiltrating T cell profiling observed in a lungcancer mouse model. *p<0.05; **p<0.01; and ***p<0.001. P-values wereobtained by comparison of each intervention group and the control group(vehicle treated). #p<0.05; ##p<0.01; ###p<0.001. P-values were obtainedby comparison of each intervention group and the anti-PD1antibody-treated group.

FIG. 9 includes charts showing the effect of Composition X-anti-PD1combinations on splenic T cell profiling observed in a lung cancer mousemodel. *p<0.05; **p<0.01; and ***p<0.001. P-values were obtained bycomparison of each intervention group and the control group (vehicletreated). #p<0.05; ##p<0.01; ###p<0.001. P-values were obtained bycomparison of each intervention group and the anti-PD1 antibody-treatedgroup.

FIG. 10 includes charts showing the PD1/PD-L1 expression profiling intumor tissues obtained from mice having lung cancer cells transplantedand treated with an anti-PD1 antibody, Composition X, or a combinationthereof. *p<0.05; **p<0.01; and ***p<0.001. P-values were obtained bycomparison of each intervention group and the control group (vehicletreated). #p<0.05; ##p<0.01; ###p<0.001. P-values were obtained bycomparison of each intervention group and the anti-PD1 antibody-treatedgroup.

FIG. 11 is a chart showing the tumor-infiltrating myeloid-derivedsuppressor cells (MDSC) profiling. *p<0.05; **p<0.01; and ***p<0.001.P-values were obtained by comparison of each intervention group and thecontrol group (vehicle treated). #p<0.05; ##p<0.01; ###p<0.001. P-valueswere obtained by comparison of each intervention group and the anti-PD1antibody-treated group.

FIG. 12 is a schematic illustration of an experimental design forstudying the effect of a combination of an anti-PD1 antibody and afermented composition (Composition X) via oral administration orintravenous injection in a colon cancer mouse model.

FIG. 13 is a photo showing sizes of tumors formed by subcutaneousinjection of colon cancer CT26 cells in mice treated with vehiclecontrol, an anti-PD1 antibody, a combinations of the anti-PD1 antibodyand a fermented composition via oral administration, or a combination ofthe anti-PD1 antibody and the fermented composition via intravenousinjection.

FIG. 14 includes charts showing the inhibitory effects of theanti-PD1/Composition X combination on tumor growth observed in a coloncancer mouse model via either oral administration or intravenousinjection.

FIG. 15 includes charts showing the effect of Composition X-anti-PD1combinations on the profiling of T cell at tumor sites observed in acolon cancer mouse model. Composition X was administered either orallyor intravenously. *p<0.05; **p<0.01; and ***p<0.001. P-values wereobtained by comparison of each intervention group and the control group(vehicle treated). #p<0.05; ##p<0.01; ###p<0.001. P-values were obtainedby comparison of each intervention group and the anti-PD1antibody-treated group.

FIG. 16 includes charts showing the effect of Composition X-anti-PD1combinations on splenic T cell profiling observed in a colon cancermouse model. Composition X was administered either orally orintravenously. *p<0.05; **p<0.01; and ***p<0.001. P-values were obtainedby comparison of each intervention group and the control group (vehicletreated). #p<0.05; ##p<0.01; ###p<0.001. P-values were obtained bycomparison of each intervention group and the anti-PD1 antibody-treatedgroup.

FIG. 17 includes charts showing the PD1/PD-L1 expression profiling intumor tissues obtained from mice having lung cancer cells transplantedand treated with an anti-PD1 antibody, Composition X, or a combinationthereof. Composition X was administered with orally or intravenously.*p<0.05; **p<0.01; and ***p<0.001. P-values were obtained by comparisonof each intervention group and the control group (vehicle treated).#p<0.05; ##p<0.01; ###p<0.001. P-values were obtained by comparison ofeach intervention group and the anti-PD1 antibody-treated group.

FIG. 18 is a chart showing the effect of an anti-PD1 antibody,Composition X, or a combination thereof on the level of NK cells(characterized as CD3e−/CD49b+). Composition X was administered withorally or intravenously. *p<0.05; **p<0.01; and ***p<0.001. P-valueswere obtained by comparison of each intervention group and the controlgroup (vehicle treated). #p<0.05; ##p<0.01; ###p<0.001. P-values wereobtained by comparison of each intervention group and the anti-PD1antibody-treated group.

FIG. 19 is a chart showing the tumor-infiltrating myeloid-derivedsuppressor cells (MDSC) profiling. *p<0.05; **p<0.01; and ***p<0.001.Composition X was administered with orally or intravenously. P-valueswere obtained by comparison of each intervention group and the controlgroup (vehicle treated). #p<0.05; ##p<0.01; ###p<0.001. P-values wereobtained by comparison of each intervention group and the anti-PD1antibody-treated group.

FIG. 20 includes charts showing the level of cytokines IL-2, IFN-γ, andIL-15 at tumor sites in mice treated with a vehicle control, anti-PD1antibody, Composition X, or a combination thereof. *p<0.05; **p<0.01;and ***p<0.001. Composition X was administered with orally orintravenously. P-values were obtained by comparison of each interventiongroup and the vehicle control group. #p<0.05; ##p<0.01; ###p<0.001.P-values were obtained by comparison of each intervention group and theanti-PD1 antibody-treated group.

FIG. 21 includes charts showing the level of cytokines IL-6, TNF-α, andIL-5 at tumor sites in mice treated with a vehicle control, anti-PD1antibody, Composition X, or a combination thereof. *p<0.05; **p<0.01;and ***p<0.001. Composition X was administered with orally orintravenously. P-values were obtained by comparison of each interventiongroup and the vehicle control group. #p<0.05; ##p<0.01; ###p<0.001.P-values were obtained by comparison of each intervention group and theanti-PD1 antibody-treated group.

FIG. 22 includes charts showing the serum level of cytokines IL-15,IL-6, IL-4 and IL-1 in mice treated with a vehicle control, anti-PD1antibody, Composition X, or a combination thereof. *p<0.05; **p<0.01;and ***p<0.001. Composition X was administered with orally orintravenously. P-values were obtained by comparison of each interventiongroup and the vehicle control group.

FIG. 23 is a diagram showing the correlation between tumor size andabundance of certain bacteria species in the intestine of mice treatedwith a vehicle, an anti-PD1 antibody, a fermented soybean composition(Composition X), or a combination of the anti-PD1 antibody andComposition X.

DETAILED DESCRIPTION OF THE INVENTION

Immune checkpoint modulators have been shown to effectively inhibitcancer cell growth in many studies. The studies provided in the presentdisclosure demonstrate, surprisingly, that fermented soybeancompositions, for example, an exemplary soybean fermented composition,Composition X described herein, successfully enhanced the anti-cancereffect of an immune checkpoint modulator (e.g., an immune checkpointinhibitor such as an anti-PD1 antibody). In some instances, the co-useof the immune checkpoint modulator and the fermented soybean compositionexhibited synergistic effects in suppressing cancer growth in an animalcancer model. Without being bound by theory, the results of this studyshow that the fermented composition may facilitate the immune checkpointinhibitor to modulate intestinal microbiota, which was reported in theart as involved in cancer development. These unexpected results indicatethat the fermented soybean composition can be co-used with one or moreimmune checkpoint modulators such as inhibitors to more effectivelytreat cancer.

Accordingly, provided herein are combined cancer therapies involving theco-use of immune checkpoint modulators and fermented soybeancompositions and kits for such uses.

I. Agents for Combined Cancer Therapy

The combined cancer therapies described herein involves the use ofimmune checkpoint modulators and fermented compositions such asfermented soybean compositions as active agents.

(a) Immune Checkpoint Modulators

Immune checkpoint proteins refer to molecules involved in a plethora ofregulatory pathways that regulate (e.g., either turning up or turningdown a signal of) the immune system to maintain self-tolerance and tomodulate the duration and amplitude of physiological immune responses inperipheral tissues in order to minimize collateral tissue damage.Typically, immune checkpoint proteins are dysregulated by cancer cells(e.g., tumors). Without wishing to be bound by any particular theory,immune checkpoint proteins can be targeted with modulators (activatorsor inhibitors) as an anti-cancer therapy, for example as described byPardoll et al., Nature Reviews Cancer, 12: 252-264, 2012.

The immune cell signaling pathways can be mediated by one or more of thefollowing exemplary receptor/ligand pairs on cells: PD1/PDL1, PD1/PDL2,CD28/B7-1 (CD80), CD28/B7-2 (CD86), CTLA4/B7-1(CD80), CILA4/B7-2(CD86),4-1BB (CD137)/4-1BBL (CD137L), ICOS/B7RP1, CD40/CD40L, Herpesvirus entrymediator (HVEM)/B- and T-lymphocyte attenuator (BTLA); OX40/OX40L,CD27/CD70, GITR/GITRL, KIR/MHC, Lymphocyte-activation gene 3 (LAG3 orCD223)/MHC, Hepatitis A virus cellular receptor 2 (HAVCR2; also known asT-cell immunoglobulin and mucin-domain containing-3 (TIM3))/TIM3 ligand,T cell immunoreceptor with Ig and ITIM domains (TIGIT)/CD96, andTIGIT/CD226. The immune cell signaling pathways can also be mediated byone or more of the following exemplary cytokines/chemokines and theircognate cell-surface receptors: interleukin 2 (IL-2)/CD122,adenosine/adenosine A2A receptor (A2AR), interleukin 6(IL-6)/IL6R(CD126), interleukin 10(IL-10)/IL-10R, interleukin15(IL-15)/IL-15R, transforming growth factor β (TGFβ)/TGFβR, andMacrophage colony-stimulating factor 1 (CSF-1)/CSF-1R. Other immunecheckpoints include, but are not limited to, KIR2DL, VISTA, HLLA2, TLIA,DNAM-1, CEACAM1, CD155, and indoleamine 2,3-dioxygenase (IDO), such asIDO1. Any of the immune checkpoint molecules described above can be atarget for anti-cancer therapy as described herein.

As used herein, “immune checkpoint modulator” refers to an agent thatalters the activity of an immune checkpoint protein (e.g., any of thosedescribed herein) in a cell relative to a control vehicle. The term“modulator” is used herein in the broadest sense, and includes anymolecule that partially or fully alters a signaling pathway regulated byone or more immune checkpoint molecules, including the signalingpathways mediated by the molecules described herein.

In some instances, an immune checkpoint modulator is an inhibitor of acheckpoint molecule that reduces, slows, halts, and/or prevents theactivity mediated by that checkpoint molecule. The term “inhibitor” isused herein in the broadest sense, and includes any molecule thatpartially or fully blocks, inhibits, or neutralizes a signaling pathwayregulated by one or more immune checkpoint molecules, including themodulatory pathways mediated by the molecules described herein. Suitableinhibitory molecules specifically include antagonist antibodies orantibody fragments, fragments or amino acid sequence variants of nativepolypeptides, peptides, antisense oligonucleotides, small organicmolecules, recombinant proteins or peptides, etc.

In other instances, an immune checkpoint modulator is an activator of acheckpoint molecule that enhances and improves the activity mediated bythat checkpoint molecule. The term “activator” is used herein in thebroadest sense, and includes any molecule that enhances a signalingpathway regulated by one or more immune checkpoint molecules, includingthe signaling pathways mediated by the molecules described herein.Suitable activators include agonistic antibodies or antibody fragments,small organic molecules, recombinant proteins or peptides, etc. In someinstances, the activator can be an agonistic antibody of a checkpointprotein, e.g., MEDI0562 (a humanized OX40 agonistic antibody), MEDI6469(a murine OX4 agonist); and MEDI6383 (an OX40 agonist).

Methods for identifying such modulators are well known in the art. Forexample, a candidate modulator can be brought in contact with a suitableimmune checkpoint target and the intensity of the signaling mediated bythat immune checkpoint can be measured by a conventional assay. Adetectable change in the signaling in the presence of the candidatemodulator as relative to a blank control indicates that the candidatemodulator possesses the regulatory activity of the immune checkpointmolecule.

An immune checkpoint modulator as described herein can be an inhibitoryagent that interferes with the signaling in an immune cell that ismediated by an immune checkpoint protein, for example, either bydecreasing transcription or translation of checkpoint protein-encodingnucleic acid, or by inhibiting or blocking the checkpoint proteinactivity, or both. Examples of immune checkpoint inhibitors include, butare not limited to, antisense polynucleotides, interfering RNAs,catalytic RNAs, RNA-DNA chimeras, checkpoint protein-specific aptamers,anti-checkpoint protein antibodies (e.g., full-length antibodies orantigen-binding fragments thereof), checkpoint-binding small molecules,checkpoint-binding peptides, and other polypeptides that specificallybind a checkpoint protein (including, but not limited to,checkpoint-binding fragments of its cognate ligand, which may optionallybe fused to one or more additional domains), such that the interactionbetween the checkpoint modulator and the checkpoint protein results in areduction or cessation of the checkpoint protein activity or expression.It will be understood by one of ordinary skill in the art that in someinstances, a checkpoint protein modulator can antagonize or neutralizeone checkpoint protein activity without affecting another checkpointprotein activity. For example, a desirable checkpoint modulator for usein certain of the methods herein can be a checkpoint modulator thatdisrupts binding interaction between the checkpoint protein and onecognate ligand without affecting or minimally affecting the interactionbetween the checkpoint protein and another cognate ligand, if any.

The immune checkpoint modulators as described herein may reduce theintensity of the signaling mediated by a checkpoint protein in immunecells by at least 20% or more, e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%,95% or above. The regulatory activity of such a modulator against thecheckpoint protein can be determined by conventional methods.

Anti-Checkpoint Antibodies

In some embodiments, the immune checkpoint modulator for use in thecombined anti-cancer therapy as described herein is an antibody thatspecifically binds to an immune checkpoint molecule and inhibits itsbioactivity, e.g., via interfering its binding to the cognate ligand. Asused herein, the term “antibody” as includes but is not limited topolyclonal, monoclonal, humanized, chimeric, Fab fragments, Fvfragments, F(ab′) fragments and F(ab′)2 fragments, as well as singlechain antibodies (scFv), fusion proteins and other synthetic proteinswhich comprise the antigen-binding site of the antibody.

Any of the antibodies that suppress the activity of an immune checkpointprotein may be prepared by conventional methods known in the art ormethods disclosed herein.

In some embodiments, antibodies specific to a target antigen (an immunecheckpoint protein such as PD1, PDL1, PDL2, CTLA4 and any othersdescribed herein) can be made by the conventional hybridoma technology.The full-length target antigen or a fragment thereof, optionally coupledto a carrier protein such as KLH, can be used to immunize a host animalfor generating antibodies binding to that antigen. The route andschedule of immunization of the host animal are generally in keepingwith established and conventional techniques for antibody stimulationand production, as further described herein. General techniques forproduction of mouse, humanized, and human antibodies are known in theart and are described herein. It is contemplated that any mammaliansubject including humans or antibody producing cells therefrom can bemanipulated to serve as the basis for production of mammalian, includinghuman hybridoma cell lines. Typically, the host animal is inoculatedintraperitoneally, intramuscularly, orally, subcutaneously,intraplantar, and/or intradermally with an amount of immunogen,including as described herein.

Hybridomas can be prepared from the lymphocytes and immortalized myelomacells using the general somatic cell hybridization technique of Kohler,B. and Milstein, C. (1975) Nature 256:495-497 or as modified by Buck, D.W., et al., In Vitro, 18:377-381 (1982). Available myeloma lines,including but not limited to X63-Ag8.653 and those from the SalkInstitute, Cell Distribution Center, San Diego, Calif., USA, may be usedin the hybridization. Generally, the technique involves fusing myelomacells and lymphoid cells using a fusogen such as polyethylene glycol, orby electrical means well known to those skilled in the art. After thefusion, the cells are separated from the fusion medium and grown in aselective growth medium, such as hypoxanthine-aminopterin-thymidine(HAT) medium, to eliminate unhybridized parent cells. Any of the mediadescribed herein, supplemented with or without serum, can be used forculturing hybridomas that secrete monoclonal antibodies. As anotheralternative to the cell fusion technique, EBV immortalized B cells maybe used to produce monoclonal antibodies specific to an immunecheckpoint protein as described herein. The hybridomas are expanded andsubcloned, if desired, and supernatants are assayed for anti-immunogenactivity by conventional immunoassay procedures (e.g., radioimmunoassay,enzyme immunoassay, or fluorescence immunoassay).

Hybridomas that may be used as source of antibodies encompass allderivatives, progeny cells of the parent hybridomas that producemonoclonal antibodies capable of interfering with a signal pathway(e.g., an inhibitory signal pathway) mediated by an immune checkpointmolecule. Hybridomas that produce such antibodies may be grown in vitroor in vivo using known procedures. The monoclonal antibodies may beisolated from the culture media or body fluids, by conventionalimmunoglobulin purification procedures such as ammonium sulfateprecipitation, gel electrophoresis, dialysis, chromatography, andultrafiltration, if desired. Undesired activity if present, can beremoved, for example, by running the preparation over adsorbents made ofthe immunogen attached to a solid phase and eluting or releasing thedesired antibodies off the immunogen. Immunization of a host animal witha target antigen or a fragment containing the target amino acid sequenceconjugated to a protein that is immunogenic in the species to beimmunized, e.g., keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, or soybean trypsin inhibitor using a bifunctional orderivatizing agent, for example maleimidobenzoyl sulfosuccinimide ester(conjugation through cysteine residues), N-hydroxysuccinimide (throughlysine residues), glutaraldehyde, succinic anhydride, SOCl, or R1N═C═NR,where R and R1 are different alkyl groups, can yield a population ofantibodies (e.g., monoclonal antibodies).

If desired, an antibody (monoclonal or polyclonal) of interest (e.g.,produced by a hybridoma) may be sequenced and the polynucleotidesequence may then be cloned into a vector for expression or propagation.The sequence encoding the antibody of interest may be maintained invector in a host cell and the host cell can then be expanded and frozenfor future use. In an alternative, the polynucleotide sequence may beused for genetic manipulation to “humanize” the antibody or to improvethe affinity (affinity maturation), or other characteristics of theantibody. For example, the constant region may be engineered to moreresemble human constant regions to avoid immune response if the antibodyis used in clinical trials and treatments in humans. It may be desirableto genetically manipulate the antibody sequence to obtain greateraffinity to the target antigen and greater efficacy in inhibiting thecheckpoint signaling pathway. It will be apparent to one of skill in theart that one or more polynucleotide changes can be made to the antibodyand still maintain its binding specificity to the target antigen.

In some examples, the anti-checkpoint antibodies described herein can befully human antibodies. Full human antibodies can be obtained by usingcommercially available animals (e.g., mice) that have been engineered toexpress specific human immunoglobulin proteins. Transgenic animals thatare designed to produce a more desirable (e.g., fully human antibodies)or more robust immune response may also be used for generation ofhumanized or human antibodies. Examples of such technology areXenoMouse™ from Amgen, Inc. (Fremont, Calif.) and HuMAb-Mouse™ and TCMouse™ from Medarex, Inc. (Princeton, N.J.). In another alternative,antibodies may be made recombinantly by phage display or yeasttechnology. See, for example, U.S. Pat. Nos. 5,565,332; 5,580,717;5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol.12:433-455, and. Alternatively, the phage display technology (McCaffertyet al., (1990) Nature 348:552-553) can be used to produce humanantibodies and antibody fragments in vitro, from immunoglobulin variable(V) domain gene repertoires from unimmunized donors.

Antigen-binding fragments of an intact antibody (full-length antibody)can be prepared via routine methods. For example, F(ab′)2 fragments canbe produced by pepsin digestion of an antibody molecule, and Fabfragments that can be generated by reducing the disulfide bridges ofF(ab′)2 fragments.

Genetically engineered antibodies, such as humanized antibodies,chimeric antibodies, single-chain antibodies, and bi-specificantibodies, can be produced via, e.g., conventional recombinanttechnology. In one example, DNA encoding a monoclonal antibody specificto a target antigen can be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the monoclonal antibodies). The hybridoma cells serve as apreferred source of such DNA. Once isolated, the DNA may be placed intoone or more expression vectors, which are then transfected into hostcells such as E. coli cells, simian COS cells, Chinese hamster ovary(CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. See, e.g., PCT Publication No. WO87/04462. The DNA can then be modified, for example, by substituting thecoding sequence for human heavy and light chain constant domains inplace of the homologous murine sequences, Morrison et al., (1984) Proc.Nat. Acad. Sci. 81:6851, or by covalently joining to the immunoglobulincoding sequence all or part of the coding sequence for anon-immunoglobulin polypeptide. In that manner, genetically engineeredantibodies, such as “chimeric” or “hybrid” antibodies; can be preparedthat have the binding specificity of a target antigen.

In other examples, the anti-checkpoint antibodies are humanizedantibodies. Humanized antibodies refer to antibodies derived fromnon-human (e.g., murine) antibodies that are specific chimericimmunoglobulins, immunoglobulin chains, or antigen-binding fragmentsthereof that contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from acomplementary determining region (CDR) of the recipient are replaced byresidues from a CDR of a non-human species (donor antibody) such asmouse, rat, or rabbit having the desired specificity, affinity, andcapacity. In some instances, Fv framework region (FR) residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Furthermore, the humanized antibody may comprise residues that are foundneither in the recipient antibody nor in the imported CDR or frameworksequences, but are included to further refine and optimize antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region or domain (Fc), typically that of a humanimmunoglobulin. Antibodies may have Fc regions modified as described inWO 99/58572. Other forms of humanized antibodies have one or more CDRs(one, two, three, four, five, six) which are altered with respect to theoriginal antibody, which are also termed one or more CDRs “derived from”one or more CDRs from the original antibody. Humanized antibodies mayalso involve affinity maturation. Methods for constructing humanizedantibodies are also well known in the art. See, e.g., Queen et al.,Proc. Natl. Acad. Sci. USA, 86:10029-10033 (1989).

In some embodiments, the checkpoint regulatory antibodies such ascheckpoint inhibitory can be chimeric antibodies. Techniques developedfor the production of “chimeric antibodies” are well known in the art.See, e.g., Morrison et al. (1984) Proc. Natl. Acad. Sci. USA 81, 6851;Neuberger et al. (1984) Nature 312, 604; and Takeda et al. (1984) Nature314:452.

In other embodiments, the checkpoint regulatory antibodies such asinhibitory antibodies can be single-chain antibody fragments (scFv). Asingle-chain antibody can be prepared via recombinant technology bylinking a nucleotide sequence coding for a heavy chain variable regionand a nucleotide sequence coding for a light chain variable region.Preferably, a flexible linker is incorporated between the two variableregions. Alternatively, techniques described for the production ofsingle chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can beadapted to produce a phage or yeast scFv library and scFv clonesspecific to an immune checkpoint protein, which can be identified fromthe library following routine procedures. Positive clones can besubjected to further screening to identify those that inhibit activityof the immune checkpoint protein.

Antibodies obtained following a method known in the art and describedherein can be characterized using methods well known in the art. Forexample, one method is to identify the epitope to which the antigenbinds, or “epitope mapping.” There are many methods known in the art formapping and characterizing the location of epitopes on proteins,including solving the crystal structure of an antibody-antigen complex,competition assays, gene fragment expression assays, and syntheticpeptide-based assays, as described, for example, in Chapter 11 of Harlowand Lane, Using Antibodies, a Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1999. In an additionalexample, epitope mapping can be used to determine the sequence to whichan antibody binds. The epitope can be a linear epitope, i.e., containedin a single stretch of amino acids, or a conformational epitope formedby a three-dimensional interaction of amino acids that may notnecessarily be contained in a single stretch (primary structure linearsequence). Peptides of varying lengths (e.g., at least 4-6 amino acidslong) can be isolated or synthesized (e.g., recombinantly) and used forbinding assays with an antibody. In another example, the epitope towhich the antibody binds can be determined in a systematic screening byusing overlapping peptides derived from the target antigen sequence anddetermining binding by the antibody. According to the gene fragmentexpression assays, the open reading frame encoding the target antigen isfragmented either randomly or by specific genetic constructions and thereactivity of the expressed fragments of the antigen with the antibodyto be tested is determined. The gene fragments may, for example, beproduced by PCR and then transcribed and translated into protein invitro, in the presence of radioactive amino acids. The binding of theantibody to the radioactively labeled antigen fragments is thendetermined by immunoprecipitation and gel electrophoresis. Certainepitopes can also be identified by using large libraries of randompeptide sequences displayed on the surface of phage particles (phagelibraries). Alternatively, a defined library of overlapping peptidefragments can be tested for binding to the test antibody in simplebinding assays. In an additional example, mutagenesis of an antigenbinding domain, domain swapping experiments and alanine scanningmutagenesis can be performed to identify residues required, sufficient,and/or necessary for epitope binding. For example, domain swappingexperiments can be performed using a mutant of a target antigen in whichvarious fragments of the immune checkpoint protein have been replaced(swapped) with sequences from a closely related, but antigenicallydistinct protein. By assessing binding of the antibody to the mutantcheckpoint polypeptide, the importance of the particular antigenfragment to antibody binding can be assessed.

Alternatively, competition assays can be performed using otherantibodies known to bind to the same antigen to determine whether anantibody binds to the same epitope as the other antibodies. Competitionassays are well known to those of skill in the art.

Nucleic acids encoding the heavy and light chain of an anti-checkpointantibody as described herein can be cloned into one expression vector,each nucleotide sequence being in operable linkage to a suitablepromoter. In one example, each of the nucleotide sequences encoding theheavy chain and light chain is in operable linkage to a distinctprompter. Alternatively, the nucleotide sequences encoding the heavychain and the light chain can be in operable linkage with a singlepromoter, such that both heavy and light chains are expressed from thesame promoter. When necessary, an internal ribosomal entry site (IRES)can be inserted between the heavy chain and light chain encodingsequences.

In some examples, the nucleotide sequences encoding the two chains ofthe antibody are cloned into two vectors, which can be introduced intothe same or different cells. When the two chains are expressed indifferent cells, each of them can be isolated from the host cellsexpressing such and the isolated heavy chains and light chains can bemixed and incubated under suitable conditions allowing for the formationof the antibody.

Generally, a nucleic acid sequence encoding one or all chains of anantibody can be cloned into a suitable expression vector in operablelinkage with a suitable promoter using methods known in the art. Forexample, the nucleotide sequence and vector can be contacted, undersuitable conditions, with a restriction enzyme to create complementaryends on each molecule that can pair with each other and be joinedtogether with a ligase. Alternatively, synthetic nucleic acid linkerscan be ligated to the termini of a gene. These synthetic linkers containnucleic acid sequences that correspond to a particular restriction sitein the vector. The selection of expression vectors/promoter would dependon the type of host cells for use in producing the antibodies.

A variety of promoters can be used for expression of the antibodiesdescribed herein, including, but not limited to, cytomegalovirus (CMV)intermediate early promoter, a viral LTR such as the Rous sarcoma virusLTR, HIV-LTR, HTLV-1 LTR, the simian virus 40 (SV40) early promoter, E.coli lac UV5 promoter, and the herpes simplex tk virus promoter.

Regulatable promoters can also be used. Such regulatable promotersinclude those using the lac repressor from E. coli as a transcriptionmodulator to regulate transcription from lac operator-bearing mammaliancell promoters [Brown, M. et al., Cell, 49:603-612 (1987)], those usingthe tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc.Natl. Acad. Sci. USA 89:5547-5551 (1992); Yao, F. et al., Human GeneTherapy, 9:1939-1950 (1998); Shockelt, P., et al., Proc. Natl. Acad.Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimer, VP16or p65 using astradiol, RU486, diphenol murislerone, or rapamycin.Inducible systems are available from Invitrogen, Clontech and Ariad.

Regulatable promoters that include a repressor with the operon can beused. In one embodiment, the lac repressor from E. coli can function asa transcriptional modulator to regulate transcription from lacoperator-bearing mammalian cell promoters [M. Brown et al., Cell,49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl.Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor(tetR) with the transcription activator (VP 16) to create atetR-mammalian cell transcription activator fusion protein, tTa (tetR-VP16), with the tetO-bearing minimal promoter derived from the humancytomegalovirus (hCMV) major immediate-early promoter to create atetR-tet operator system to control gene expression in mammalian cells.In one embodiment, a tetracycline inducible switch is used. Thetetracycline repressor (tetR) alone, rather than the tetR-mammalian celltranscription factor fusion derivatives can function as potenttrans-modulator to regulate gene expression in mammalian cells when thetetracycline operator is properly positioned downstream for the TATAelement of the CMVIE promoter (Yao et al., Human Gene Therapy). Oneparticular advantage of this tetracycline inducible switch is that itdoes not require the use of a tetracycline repressor-mammalian cellstransactivator or repressor fusion protein, which in some instances canbe toxic to cells (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551(1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526(1995)), to achieve its regulatable effects.

Additionally, the vector can contain, for example, some or all of thefollowing: a selectable marker gene, such as the neomycin gene forselection of stable or transient transfectants in mammalian cells;enhancer/promoter sequences from the immediate early gene of human CMVfor high levels of transcription; transcription termination and RNAprocessing signals from SV40 for mRNA stability; SV40 polyoma origins ofreplication and ColE1 for proper episomal replication; internal ribosomebinding sites (IRESes), versatile multiple cloning sites; and T7 and SP6RNA promoters for in vitro transcription of sense and antisense RNA.Suitable vectors and methods for producing vectors containing transgenesare well known and available in the art.

Examples of polyadenylation signals useful to practice the methodsdescribed herein include, but are not limited to, human collagen Ipolyadenylation signal, human collagen II polyadenylation signal, andSV40 polyadenylation signal.

One or more vectors (e.g., expression vectors) comprising nucleic acidsencoding any of the antibodies may be introduced into suitable hostcells for producing the antibodies. The host cells can be cultured undersuitable conditions for expression of the antibody or any polypeptidechain thereof. Such antibodies or polypeptide chains thereof can berecovered by the cultured cells (e.g., from the cells or the culturesupernatant) via a conventional method, e.g., affinity purification. Ifnecessary, polypeptide chains of the antibody can be incubated undersuitable conditions for a suitable period of time allowing forproduction of the antibody.

In some embodiments, methods for preparing an antibody described hereininvolve a recombinant expression vector that encodes both the heavychain and the light chain of an anti-checkpoint antibody, as alsodescribed herein. The recombinant expression vector can be introducedinto a suitable host cell (e.g., a dhfr-CHO cell) by a conventionalmethod, e.g, calcium phosphate-mediated transfection. Positivetransformant host cells can be selected and cultured under suitableconditions allowing for the expression of the two polypeptide chainsthat form the antibody, which can be recovered from the cells or fromthe culture medium. When necessary, the two chains recovered from thehost cells can be incubated under suitable conditions allowing for theformation of the antibody.

In one example, two recombinant expression vectors are provided, oneencoding the heavy chain of the anti-checkpoint antibody and the otherencoding the light chain of the anti-checkpoint antibody. Both of thetwo recombinant expression vectors can be introduced into a suitablehost cell (e.g., dhfr-CHO cell) by a conventional method, e.g., calciumphosphate-mediated transfection. Alternatively, each of the expressionvectors can be introduced into a suitable host cells. Positivetransformants can be selected and cultured under suitable conditionsallowing for the expression of the polypeptide chains of the antibody.When the two expression vectors are introduced into the same host cells,the antibody produced therein can be recovered from the host cells orfrom the culture medium. If necessary, the polypeptide chains can berecovered from the host cells or from the culture medium and thenincubated under suitable conditions allowing for formation of theantibody. When the two expression vectors are introduced into differenthost cells, each of them can be recovered from the corresponding hostcells or from the corresponding culture media. The two polypeptidechains can then be incubated under suitable conditions for formation ofthe antibody.

Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recovery of the antibodiesfrom the culture medium. For example, some antibodies can be isolated byaffinity chromatography with a Protein A or Protein G coupled matrix.

Any of the nucleic acids encoding the heavy chain, the light chain, orboth of an anti-checkpoint polypeptide antibody as described herein,vectors (e.g., expression vectors) containing such; and host cellscomprising the vectors are within the scope of the present disclosure.

Other Immune Checkpoint Modulators

In some embodiments, the checkpoint modulator is an interfering RNA suchas a small interfering RNA (siRNA) short hairpin RNA (shRNA). In someembodiments, the checkpoint modulator is a small interfering RNA (siRNA)that binds to the mRNA of the checkpoint protein and blocks itstranslation or degrades the mRNA via RNA interference. Exemplary smallinterfering RNAs are described by Hannon et al., Nature, 418 (6894):244-51 (2002); Brummelkamp et al., Science 21 (2002); and Sui et al.,Proc. Natl Acad. Sci. USA 99, 5515-5520 (2002). RNA interference (RNAi)is the process of sequence-specific post-transcriptional gene silencingin animals initiated by double-stranded (dsRNA) that is homologous insequence to the silenced gene. siRNAs are generally RNA duplexes witheach strand being 20-25 (such as 19-21) base pairs in length. In someembodiments, the checkpoint modulator is a short hairpin RNA (shRNA)that is complementary to a nucleic acid (e.g., a mRNA) of the checkpointprotein or a fragment thereof. An shRNA typically contains of a stem of19-29 base pairs, a loop of at least 4 nucleotides (nt), and optionallya dinucleotide overhang at the 3′ end. Expression of shRNA in a subjectcan be obtained by delivery of a vector (e.g., a plasmid or viral orbacterial vectors) encoding the shRNA. siRNAs and shRNAs may be designedusing any method known in the art or commercially available (see, e.g.,products available from Dharmacon and Life Technologies). An siRNA mayalso comprise one or more chemical modifications, such as a basemodification and/or a bond modification to at least improve itsstability and binding affinity to the target mRNA.

In some embodiments, the checkpoint modulator is an antisenseoligonucleotide that is complementary to a nucleic acid (e.g., an mRNA)of the checkpoint protein. Antisense oligonucleotides are generallysingle-stranded nucleic acids (a DNA, RNA, or hybrid RNA-DNA molecule),which are complementary to a target nucleic acid sequence, such as aportion of the mRNA of the checkpoint protein. By binding to the targetsequence, an RNA-RNA, a DNA-DNA, or RNA-DNA duplex is formed, therebyinhibiting the function or level of the target nucleic acid, such as byblocking the transcription, processing, poly(A) addition, replication,translation, or promoting modulatory mechanisms of the cells, such aspromoting mRNA degradation. In some embodiments, an antisenseoligonucleotide is 10 to 40, 12 to 35, or 15 to 35 bases in length, orany integer in between. An antisense oligonucleotide can comprise one ormore modified bases, such as 2-Aminopurine, 2,6-Diaminopurine(2-Amino-dA), 5-Bromo dU, 5-Methyl dC, deoxylnosine, Locked Nucleic Acid(LNA), 5-Nitroindole, 2′-O-Methyl bases, Hydroxmethyl dC, 2′ Fluorobases. An antisense oligonucleotide can comprise one or more modifiedbonds, such as a phosphorothioate bond.

In some embodiments, the checkpoint modulator is a ribozyme that iscomplementary to a nucleic acid (e.g., an mRNA) of the checkpointprotein and cleaves the nucleic acid. Ribozymes are RNA or RNA-proteincomplexes that cleave nucleic acids in a site-specific fashion.Ribozymes have specific catalytic domains that possess endonucleaseactivity. The ribozymes of the present disclosure may be syntheticribozymes, such as those described in U.S. Pat. No. 5,254,678. Thesesynthetic ribozymes have separate hybridizing regions and catalyticregions; therefore, the hybridizing regions can be designed to recognizea target sequence, such as a sequence as described herein correspondingto a checkpoint protein.

siRNAs, shRNAs, ribozymes, and antisense oligonucleotides as describedherein may be complementary to a nucleic acid (e.g., a mRNA) of acheckpoint protein, or a portion thereof. It is to be understood thatcomplementarity includes 100% complementarity but does not necessarilyexclude mismatches at one or more locations, resulting in, e.g., atleast 80%, at least 90%, at least 95%, at least 98%, or at least 99%complementarity.

When applicable, the checkpoint modulator can be expressed from avector, which may be used for delivering the checkpoint modulator into asubject who needs an anti-cancer therapy. A “vector”, as used herein isany vehicle capable of facilitating the transfer of a checkpointmodulator (e.g., a shRNA, siRNA, ribozyme, antisense oligonucleotide,protein, peptide, or antibody) to a cell in the subject, such as a cellexpressing the checkpoint protein. In general, vectors include, but arenot limited to, plasmids, phagemids, viruses, and other vehicles derivedfrom viral or bacterial sources that have been manipulated by theinsertion or incorporation of a sequence encoding a checkpointmodulator. Viral vectors include, but are not limited to nucleic acidsequences from the following viruses: retrovirus; lentivirus;adenovirus; adeno-associated virus; SV40-type viruses; polyoma viruses;Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus;polio virus. One can readily employ other vectors not named but known tothe art.

Viral vectors may be based on non-cytopathic eukaryotic viruses in whichnonessential genes have been replaced with a sequence encoding acheckpoint modulator. Non-cytopathic viruses include retroviruses (e.g.,lentivirus), the life cycle of which involves reverse transcription ofgenomic viral RNA into DNA with subsequent proviral integration intohost cellular DNA. Retroviruses have been approved for human genetherapy trials. Most useful are those retroviruses that arereplication-deficient (i.e., capable of directing synthesis of thedesired proteins, but incapable of manufacturing an infectiousparticle). Such genetically altered retroviral expression vectors havegeneral utility for the high-efficiency transduction of genes in vivo.Standard protocols for producing replication-deficient retroviruses(including the steps of incorporation of exogenous genetic material intoa plasmid, transfection of a packaging cell lined with plasmid,production of recombinant retroviruses by the packaging cell line,collection of viral particles from tissue culture media, and infectionof the target cells with viral particles) are known in the art.

Other viral vectors include adeno-viruses and adeno-associated viruses,which are double-stranded DNA viruses that have also been approved forhuman use in gene therapy. The adeno-associated virus can be engineeredto be replication deficient and is capable of infecting a wide range ofcell types and species.

Other vectors include plasmid vectors. Plasmid vectors have beenextensively described in the art and are well known to those of skill inthe art. See, e.g., Sambrook et al. Molecular Cloning: A LaboratoryManual. Cold Spring Harbor Laboratory Press; 4th edition (Jun. 15,2012). Exemplary plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40,and pBlueScript. Other plasmids are well known to those of ordinaryskill in the art. Additionally, plasmids may be custom designed usingrestriction enzymes and ligation reactions to remove and add specificfragments of DNA, such as a sequence encoding an immune checkpointmodulator.

In some embodiments, a checkpoint modulator nucleic acid sequence can beunder the control of a heterologous regulatory region, e.g., aheterologous promoter. The promoter can be, e.g., a ubiquitous promotersuch as a CMV promoter, ActB promoter, or Ubiquitin B promoter. Thepromoter can also be a tissue-specific promoter or synthetic promoter.Promoters are well known in the art and commercially available (see,e.g., products available from InvivoGen).

In some embodiments, the checkpoint modulator can be a non-antibodypeptide or protein. The peptide or protein may comprise an amino acidsequence that interferes with the checkpoint signaling, such as bycompeting with a natural ligand for the involved checkpoint protein.Proteins and peptides may be designed using any method known in the art,e.g., by screening libraries of proteins or peptides for binding to acheckpoint protein or inhibition of the checkpoint protein from bindingto a ligand. In some examples, the non-antibody peptide or proteinmodulator can be a soluble receptor, for example, an Fc-fusion solublereceptor.

In some embodiments, a checkpoint modulator can be a fragment or variantof the checkpoint protein itself, e.g., a fragment that binds a cognateligand thereof but does not transmit the corresponding signaling. Acheckpoint modulator of this type can be a dominant negative modulator.

In some embodiments, the checkpoint modulator is a small molecule, suchas a small organic molecule, which typically has a molecular weight lessthan 5,000 kDa. Suitable small molecules include those that bind to animmune checkpoint molecule, or a fragment thereof, and may be identifiedby methods such as screening large libraries of compounds(Beck-Sickinger & Weber (2001) Combinational Strategies in Biology andChemistry (John Wiley & Sons, Chichester, Sussex); by structure-activityrelationship by nuclear magnetic resonance (Shuker et al (1996)“Discovering high-affinity ligands for proteins: SAR by NMR. Science274: 1531-1534); encoded self-assembling chemical libraries Melkko et al(2004) “Encoded self-assembling chemical libraries.” Nature Biotechnol.22: 568-574); DNA-templated chemistry (Gartner et al (2004) “DNA-templated organic synthesis and selection of a library of macrocycles.Science 305: 1601-1605); dynamic combinatorial chemistry (Ramstrom &Lehn (2002) “Drug discovery by dynamic combinatorial libraries.” NatureRev. Drug Discov. 1: 26-36); tethering (Arkin & Wells (2004)“Small-molecule modulators of protein-protein interactions: progressingtowards the dream. Nature Rev. Drug Discov. 3: 301-317); and speedscreen (Muckenschnabel et al (2004) “SpeedScreen: label-free liquidchromatography-mass spectrometry-based high-throughput screening for thediscovery of orphan protein ligands.” Anal. Biochem. 324: 241-249).Typically, small molecules will have a dissociation constant for animmune checkpoint protein in the nanomolar range.

Pharmaceutical Compositions Containing Checkpoint Modulators

Any of the immune checkpoint modulators, e.g., antibodies, the encodingnucleic acids or nucleic acid sets, vectors comprising such, siRNAs,antisense RNAs, and vectors carrying such, and small moleculemodulators, can be mixed with a pharmaceutically acceptable carrier(excipient) to form a pharmaceutical composition for use in treating atarget disease (e.g., cancer). “Acceptable” means that the carrier mustbe compatible with the active ingredient of the composition (andpreferably, capable of stabilizing the active ingredient) and notdeleterious to the subject to be treated. Pharmaceutically acceptableexcipients (carriers) including buffers, which are well known in theart. See, e.g., Remington: The Science and Practice of Pharmacy 20th Ed.(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

The pharmaceutical compositions to be used in the present methods cancomprise pharmaceutically acceptable carriers, excipients, orstabilizers in the form of lyophilized formulations or aqueoussolutions. (Remington: The Science and Practice of Pharmacy 20th Ed.(2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover). Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations used, and may comprise buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrans; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

In some examples, the pharmaceutical composition described hereincomprises liposomes containing the antibodies (or the encoding nucleicacids) which can be prepared by methods known in the art, such asdescribed in Epstein, et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985);Hwang, et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Pat.Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation timeare disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomescan be generated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes areextruded through filters of defined pore size to yield liposomes withthe desired diameter.

The antibodies, or the encoding nucleic acid(s), may also be entrappedin microcapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are known in theart, see, e.g., Remington, The Science and Practice of Pharmacy 20th Ed.Mack Publishing (2000).

In other examples, the pharmaceutical composition described herein canbe formulated in sustained-release format. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), sucrose acetate isobutyrate, andpoly-D-(−)-3-hydroxybutyric acid.

The pharmaceutical compositions to be used for in vivo administrationmust be sterile. This is readily accomplished by, for example,filtration through sterile filtration membranes. Therapeutic antibodycompositions are generally placed into a container having a sterileaccess port, for example, an intravenous solution bag or vial having astopper pierceable by a hypodermic injection needle.

The pharmaceutical compositions described herein can be in unit dosageforms such as tablets, pills, capsules, powders, granules, solutions orsuspensions, or suppositories, for oral, parenteral or rectaladministration, or administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient can be mixed with a pharmaceutical carrier, e.g.,conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g., water, toform a solid preformulation composition containing a homogeneous mixtureof a compound of the present invention, or a non-toxic pharmaceuticallyacceptable salt thereof. When referring to these preformulationcompositions as homogeneous, it is meant that the active ingredient isdispersed evenly throughout the composition so that the composition maybe readily subdivided into equally effective unit dosage forms such astablets, pills and capsules. This solid preformulation composition isthen subdivided into unit dosage forms of the type described abovecontaining from 0.1 to about 500 mg of the active ingredient of thepresent invention. The tablets or pills of the novel composition can becoated or otherwise compounded to provide a dosage form affording theadvantage of prolonged action. For example, the tablet or pill cancomprise an inner dosage and an outer dosage component, the latter beingin the form of an envelope over the former. The two components can beseparated by an enteric layer that serves to resist disintegration inthe stomach and permits the inner component to pass intact into theduodenum or to be delayed in release. A variety of materials can be usedfor such enteric layers or coatings, such materials including a numberof polymeric acids and mixtures of polymeric acids with such materialsas shellac, cetyl alcohol and cellulose acetate.

Suitable surface-active agents include, in particular, non-ionic agents,such as polyoxyethylenesorbitans (e.g., Tween™ 20, 40, 60, 80 or 85) andother sorbitans (e.g., Span™20, 40, 60, 80 or 85). Compositions with asurface-active agent will conveniently comprise between 0.05 and 5%surface-active agent, and can be between 0.1 and 2.5%. It will beappreciated that other ingredients may be added, for example mannitol orother pharmaceutically acceptable vehicles, if necessary.

Suitable emulsions may be prepared using commercially available fatemulsions, such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ andLipiphysan™. The active ingredient may be either dissolved in apre-mixed emulsion composition or alternatively it may be dissolved inan oil (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil,corn oil or almond oil) and an emulsion formed upon mixing with aphospholipid (e.g., egg phospholipids, soybean phospholipids or soybeanlecithin) and water. It will be appreciated that other ingredients maybe added, for example glycerol or glucose, to adjust the tonicity of theemulsion. Suitable emulsions will typically contain up to 20% oil, forexample, between 5 and 20%.

The emulsion compositions can be those prepared by mixing an antibodywith Intralipid™ or the components thereof (soybean oil, eggphospholipids, glycerol and water).

Pharmaceutical compositions for inhalation or insufflation includesolutions and suspensions in pharmaceutically acceptable, aqueous ororganic solvents, or mixtures thereof, and powders. The liquid or solidcompositions may contain suitable pharmaceutically acceptable excipientsas set out above. In some embodiments, the compositions are administeredby the oral or nasal respiratory route for local or systemic effect.

Compositions in preferably sterile pharmaceutically acceptable solventsmay be nebulised by use of gases. Nebulised solutions may be breatheddirectly from the nebulising device or the nebulising device may beattached to a face mask, tent or intermittent positive pressurebreathing machine. Solution, suspension or powder compositions may beadministered, preferably orally or nasally, from devices which deliverthe formulation in an appropriate manner.

(b) Fermented Compositions of Legume Plants

Fermentation is a metabolic process, in which microorganisms such asyeast and bacteria convert carbohydrates to acids (e.g., organic acidssuch as lactic acids), alcohols (e.g., ethanol) and/or other metabolitesunder anaerobic conditions. The fermented composition for use in thecombined anti-cancer therapy as described herein is a fermentationproduct of one or more legume plants by one or more suitablemicroorganisms, for example a population of symbiotic microbiota. Such afermented composition may comprise multiple metabolites derived fromfermenting a suitable starting material, for example, a legume plant, aportion thereof (e.g., leave, fruit, seed, etc.), or an extract thereof,by one or more suitable microorganisms, via, e.g., a conventionalfermentation process. Suitable microorganisms include, but are notlimited to, yeast and lactobacillus. See, e.g., US20060251748, therelevant disclosures of which are incorporated by reference herein forthe purposes or subject matter referenced herein. In some embodiments,the fermented composition described herein may comprise one or more ofmetabolites such as lactic acid, acetic acid, and 3-aminoisobutyricacid. For example, the fermented composition may comprise about 5-20% byweight lactic acid (e.g., 5-10%, 10-20%, 5-15%, or 15-20% by weight).Alternative or in addition, the fermented composition may comprise lessthan 5% by weight acetic acid, 3-aminoisobutyric acid, or both (e.g.,1-5%, 0.5-5%, 1-3%, 0.5-3%, or 3-5% by weight).

Exemplary legume plants include, but are not limited to, beans, peas,alfalfa, red clover, fava, vetch, and cowpeas. In some embodiments, thefermented composition for use in the combined anti-cancer therapydisclosed herein is a fermented soybean composition, which comprisesmultiple metabolites that are generated via fermentation of soybeans oran extract thereof (e.g., an aqueous extract) by one or more suitablemicroorganisms such as yeast and lactobacillus. As well known in theart, such metabolites may be generated via alternative processes, whichare also within the scope of the present disclosure.

Any of the fermented compositions described herein, such as fermentedsoybean compositions, may be prepared by fermenting a legume plant, aportion thereof, or an extract thereof by one or more suitablemicroorganisms such as those described herein under suitable conditionsallowing for fermentation of the components of the legume plant toproduce metabolites thereof. For example, a fermented composition may beprepared by culturing the one or more suitable microorganisms in amedium comprising an aqueous extract of a legume plant of a portionthereof under suitable conditions (e.g., a suitable temperature of, forexample, 20-45° C. (e.g., 20-25° C., 20-30° C., 25-30° C., 25 to 35° C.,30-45° C., or 30-40° C.) for a suitable period of time (e.g., 2-10 dayssuch as 2-5 days, 4-8 days, and 5-10 days). The one or moremicroorganisms for use in preparing the fermented composition can besymbiotic, which refers to diverse organisms that live together andbenefit each other. The supernatant of the culture can then becollected, filtered to remove solid components, and sterilized. Whennecessary, the supernatant may be concentrated by a conventional method(e.g., dialysis) to produce a concentrated fermented compositionsolution.

In one example, the fermented composition is a fermented soybeancomposition, which can be prepared by obtaining an aqueous extract ofsoybeans via a conventional method, fermenting the aqueous extract witha mixture of at least one Lactobacillus and at least one Saccharomycesunder suitable conditions for a suitable period to form a fermentedliquid, collecting, filtering, and sterilizing the fermented liquid, andremoving water from the sterilized and fermented liquid to form aconcentrated fermented soybean composition.

In some examples, the fermented composition is in liquid form. In otherexamples, the fermented composition may be in dry form, for example,powder, which can be prepared by routine practice (e.g., spray drying orfreeze drying).

Methods for preparing fermented compositions are also described in,e.g., U.S. Pat. Nos. 6,685,973, 6,855,350, 6,733,801, and US20120058104,the relevant disclosures of each of which are incorporated by referenceherein for the purposes or subject matter referenced herein.

Any of the fermented compositions described herein may be formulated aspharmaceutical composition following the descriptions provided herein.

In other embodiments, the fermented compositions may be formulated ashealthy food products, nutraceutical products or medical food products,which can be any kinds of liquid and solid/semi-solid materials toimprove immunity in cancer patients, including those who have beenundergone or is being treated with an immune checkpoint modulator. Thehealth food product may be a food product (e.g., tea-based beverages,juice, soft drinks, coffee, milk, jelly, cookies, cereals, chocolates,snack bars, herbal extracts, dairy products (e.g., ice cream, andyogurt)), a food/dietary supplement, or a nutraceutical formulation.

The health food product, nutraceutical products, and/or medical foodproducts described herein, containing any of the fermented compositionssuch as a fermented soybean composition, may comprise one or more ediblecarriers, which confer one or more of the benefits to the fermentedcomposition as described herein. Examples of edible carriers includestarch, cyclodextrin, maltodextrin, methylcellulose, carbonmethoxycellulose, xanthan gum, and aqueous solutions thereof. Other examplesinclude solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, stabilizers, gels, binders,excipients, disintegration agents, lubricants, sweetening agents,flavoring agents, dyes, such like materials and combinations thereof, aswould be known to one of ordinary skill in the art.

In some examples, the fermented composition can be a nutraceuticalcomposition, which refers to compositions containing components fromfood sources and conferring extra health benefits in addition to thebasic nutritional value found in foods. A nutraceutical composition asdescribed herein comprises the fermented composition and additionalingredients and supplements that promote good health and/or enhancestability and bioactivity of the fermented composition.

The actions of nutraceutical compositions may be fast or/and short-termor may help achieve long-term health objectives as those describedherein, e.g., improving immunity in a subject against cancer andenhancing the anti-cancer effect of an immune checkpoint modulator. Thenutraceutical compositions may be contained in an edible material, forexample, as a dietary supplement or a pharmaceutical formulation. As adietary supplement, additional nutrients, such as vitamins, minerals oramino acids may be included. The composition can also be a drink or afood product, e.g., tea, soft drink, juice, milk, coffee, cookie,cereal, chocolate, and snack bar. If desired, the composition can besweetened by adding a sweetener such as sorbitol, maltitol, hydrogenatedglucose syrup and hydrogenated starch hydrolyzate, high fructose cornsyrup, cane sugar, beet sugar, pectin, or sucralose.

In some embodiments, the fermented composition described herein, such asa fermented soybean composition, may be formulated as medical foodproducts. A medical food product is a food product formulated to beconsumed or administered enterally. Such a food product is usually usedunder the supervision of a physician for the specific dietary managementof a target disease, such as those described herein. In some instances,such a medical food composition is specially formulated and processed(as opposed to a naturally occurring foodstuff used in a natural state)for a patient in need of the treatment (e.g., human patients who sufferfrom cancer). In some examples, a medical food composition describedherein is not one of those that would be simply recommended by aphysician as part of an overall diet to manage the symptoms or reducethe risk of a disease or condition.

Any of the medical food compositions described herein, comprising thefermented composition as described herein, can be in the form of aliquid solution; powder, bar, wafer, a suspension in an appropriateliquid or in a suitable emulsion, as detailed below. The at least onecarrier, which can be either naturally-occurring or synthetic(non-naturally occurring), would confer one or more benefits to thefermented composition, for example, stability, bioavailability, and/orbioactivity. Any of the carriers described herein may be used for makingthe medical food composition. In some embodiments, the medical foodcomposition may further comprise one or more additional ingredientsselected from the group including, but not limited to natural flavors,artificial flavors, major trace and ultra-trace minerals, minerals,vitamins, oats, nuts, spices, milk, egg, salt, flour, lecithin, xanthangum and/or sweetening agents. The medical food composition may be placedin a suitable container, which may further comprise at least anadditional therapeutic agent such as those described herein.

The fermented compositions disclosed herein can be in the form of asolution. For example, the fermented composition can be provided in amedium, such as a buffer, a solvent, a diluent, an inert carrier, anoil, or a creme. In some examples, the fermented composition is presentin an aqueous solution that optionally contains a non-aqueousco-solvent, such as an alcohol. The fermented composition can also be inthe form of powder, paste, jelly, capsule, or tablet. Lactose and cornstarch are commonly used as diluents for capsules and as carriers fortablets. Lubricating agents, such as magnesium stearate, are typicallyadded to form tablets.

The fermented compositions may be formulated for a suitableadministration route, for example, oral administration. For oraladministration, the composition can take the form of, for example,tablets or capsules, prepared by conventional means with acceptableexcipients such as binding agents (for example, pregelatinised maizestarch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers(for example, lactose, microcrystalline cellulose or calcium hydrogenphosphate); lubricants (for example, magnesium stearate, talc orsilica); disintegrants (for example, potato starch or sodium starchglycolate); or wetting agents (for example, sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Also includedare bars and other chewable formulations.

In some examples, the fermented composition can be in a liquid form andthe one or more edible carriers can be a solvent or dispersion mediumcomprising but not limited to, ethanol, polyol (e.g., glycerol,propylene glycol, liquid polyethylene glycol), lipids (e.g.,triglycerides, vegetable oils, liposomes) or combinations thereof. Theproper fluidity can be maintained, for example, by the use of a coating,such as lecithin; by the maintenance of the required particle size bydispersion in carriers such as, for example liquid polyol or lipids; bythe use of surfactants such as, for example hydroxypropylcellulose; orcombinations thereof. In many cases, it will be advisable to include anisotonic agent, such as, for example, sugars, sodium chloride orcombinations thereof.

Liquid preparations for oral administration can take the form of, forexample, solutions, syrups or suspensions, or they can be presented as adry product for constitution with water or other suitable vehicle beforeuse. In one embodiment, the liquid preparations can be formulated foradministration with fruit juice. Such liquid preparations can beprepared by conventional means with pharmaceutically acceptableadditives such as suspending agents (for example, sorbitol syrup,cellulose derivatives or hydrogenated edible fats); emulsifying agents(for example, lecithin or acacia); non-aqueous vehicles (for example,almond oil, oily esters, ethyl alcohol or fractionated vegetable oils);and preservatives (for example, methyl or propyl-p-hydroxybenzoates,benzoate or sorbate).

In some embodiments, the fermented composition and the immune checkpointmodulator as described herein may be formulated in one composition.

II. Kits for Treating Cancer

The present disclosure also provides kits for use in treating cancerwith any of the immune checkpoint modulators described herein and any ofthe fermented composition (e.g., fermented soybean composition) alsodescribed herein. Such kits can include one or more containerscomprising an immune checkpoint modulator and a fermented composition.For example, the kit may include an anti-PD1 antibody or its encodingnucleic acid, and a fermented soybean composition.

In some embodiments, the kit can comprise instructions for use inaccordance with any of the methods described herein. The includedinstructions can comprise, for example, a description of administrationof an immune checkpoint modulator and a fermented composition to treat,delay the onset, or alleviate a proliferative disease such as cancer(e.g., colon cancer or lung cancer) according to any of the methodsdescribed herein. The kit may further comprise a description ofselecting an individual suitable for treatment based on identifyingwhether that individual has the disease. In still other embodiments, theinstructions comprise a description of administering one or more agentsof the disclosure to an individual at risk of the disease.

The instructions relating to the use of an immune checkpoint modulatorsuch as an anti-checkpoint antibody in combination with a fermentedcomposition (e.g., a fermented soybean composition) generally includeinformation as to dosage, dosing schedule, and route of administrationfor the intended treatment. The containers may be unit doses, bulkpackages (e.g., multi-dose packages) or sub-unit doses. Instructionssupplied in the kits of the invention are typically written instructionson a label or package insert (e.g., a paper sheet included in the kit),but machine-readable instructions (e.g., instructions carried on amagnetic or optical storage disk) are also acceptable.

The label or package insert may indicate that the composition is usedfor treating, alleviating and/or delaying the onset of cancer.Instructions may be provided for practicing any of the methods describedherein.

The kits of this invention are in suitable packaging. Suitable packagingincludes, but is not limited to, vials, bottles, jars, flexiblepackaging (e.g., sealed Mylar or plastic bags), and the like. Alsocontemplated are packages for use in combination with a specific device,such as an inhaler, nasal administration device (e.g., an atomizer) oran infusion device such as a minipump. A kit may have a sterile accessport (for example the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thecontainer may also have a sterile access port (for example the containermay be an intravenous solution bag or a vial having a stopper pierceableby a hypodermic injection needle).

Kits may optionally provide additional components such as buffers andinterpretive information. Normally, the kit comprises a container and alabel or package insert(s) on or associated with the container. In someembodiments, the invention provides articles of manufacture comprisingcontents of the kits described above.

Combined Therapy

Provided herein are combined cancer therapies using a combination of animmune checkpoint modulator and a fermented composition as describedherein. The term combination therapy, as used herein, embracesadministration of these agents (e.g., an immune checkpoint modulator anda fermented composition) in a sequential manner, that is, wherein eachtherapeutic agent is administered at a different time, as well asadministration of these therapeutic agents, or at least two of theagents, in a substantially simultaneous manner. Sequential orsubstantially simultaneous administration of each agent can be affectedby any appropriate route including, but not limited to, oral routes,intravenous routes, intramuscular, subcutaneous routes, and directabsorption through mucous membrane tissues. The agents can beadministered by the same route or by different routes. For example, afirst agent (e.g., a fermented composition) can be administered orally,and a second agent (e.g., an anti-checkpoint antibody such as ananti-PD1 antibody) can be administered intravenously. Further, an agentof the combination selected may be administered by intravenous injectionwhile the other agents of the combination may be administered orally.Alternatively, for example, two or more of the agents may beadministered by intravenous or subcutaneous injection.

As used herein, the term “sequential” means, unless otherwise specified,characterized by a regular sequence or order, e.g., if a dosage regimenincludes the administration of an immune checkpoint modulator such as anantibody and a fermented composition, a sequential dosage regimen couldinclude administration of the immune checkpoint modulator before,simultaneously, substantially simultaneously, or after administration ofthe fermented composition, but both agents will be administered in aregular sequence or order. The term “separate” means, unless otherwisespecified, to keep apart one from the other. The term “simultaneously”means, unless otherwise specified, happening or done at the same time,i.e., the agents of the present disclosure are administered at the sametime. The term “substantially simultaneously” means that the agents areadministered within minutes of each other (e.g., within 10 minutes ofeach other) and intends to embrace joint administration as well asconsecutive administration, but if the administration is consecutive itis separated in time for only a short period (e.g., the time it wouldtake a medical practitioner to administer two compounds separately). Asused herein, concurrent administration and substantially simultaneousadministration are used interchangeably. Sequential administrationrefers to temporally separated administration of the agents describedherein.

Combination therapy can also embrace the administration of the agentsdescribed herein (e.g., an immune checkpoint modulator and a fermentedcomposition) in further combination with other biologically activeingredients (e.g., a different antineoplastic agent) and non-drugtherapies (e.g., surgery or radiation treatment). Where the combinationtherapy further comprises radiation treatment, the radiation treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andradiation treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the radiation treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

It should be appreciated that any combination of an immune checkpointmodulator, and a fermented composition as described herein may be usedin any sequence for treating cancer. The combinations described hereinmay be selected on the basis of a number of factors, which include, butare not limited to, the effectiveness of inhibiting or preventing cancerprogression, the effectiveness for mitigating the side effects ofanother agent of the combination, or the effectiveness of mitigatingcancer related symptoms. For example, a combined therapy describedherein may reduce any of the side effects associated with eachindividual members of the combination. Some examples are provided in thebelow tables. For example, the fermented composition may be used duringthe course of a treatment involving an immune checkpoint modulator(e.g., an anti-PD1 antibody) on a daily basis.

Any of the combinations of an immune checkpoint modulator and afermented composition, as described herein, are useful for treatingcancer. The term “cancer” as used herein refers to a medical conditionmediated by neoplastic or malignant cell group, proliferation, ormetastasis, including solid cancers and non-solid cancers. Examples ofcancer include but are not limited to, lung cancer, kidney cancer,gastric cancer, breast cancer, brain cancer, prostate cancer,hepatocellular cancer, pancreatic cancer, cervical cancer, ovariancancer, liver cancer, bladder cancer, cancer of the urinary tract,thyroid cancer, melanoma, head and neck cancer, colon cancer, leukemia,lymphomas, skin cancer, stomach cancer, esophageal cancer, myelomas,rectal cancer, bone cancer, uterine cancer, prostate cancer, andhematological malignancy.

To practice the method disclosed herein, an effective amount of theanti-cancer agent combination as described herein can be administered toa subject (e.g., a human) in need of the treatment via a suitable route,such as intravenous administration, e.g., as a bolus or by continuousinfusion over a period of time, by intramuscular, intraperitoneal,intracerebrospinal, subcutaneous, intra-articular, intrasynovial,intrathecal, oral, inhalation or topical routes. Commercially availablenebulizers for liquid formulations, including jet nebulizers andultrasonic nebulizers are useful for administration. Liquid formulationscan be directly nebulized and lyophilized powder can be nebulized afterreconstitution. Alternatively, the immune checkpoint modulators asdescribed herein (e.g., antibodies) and/or the fermented composition canbe aerosolized using a fluorocarbon formulation and a metered doseinhaler, or inhaled as a lyophilized and milled powder.

The terms “subject,” “individual,” and “patient” are usedinterchangeably herein and refer to a mammal being assessed fortreatment and/or being treated. Subjects may be human, but also includeother mammals, particularly those mammals useful as laboratory modelsfor human disease, e.g. mouse, rat, rabbit, dog, etc.

A human subject who needs the treatment may be a human patient having,at risk for, or suspected of having a target disease/disorder, such as aproliferative disease (e.g., cancer). A subject having a target diseaseor disorder can be identified by routine medical examination, e.g.,laboratory tests, organ functional tests, CT scans, or ultrasounds. Asubject suspected of having any of such target disease/disorder mightshow one or more symptoms of the disease/disorder. A subject at risk forthe disease/disorder can be a subject having one or more of the riskfactors for that disease/disorder. The methods and compositionsdescribed herein may be used to treat any proliferative disease ordisorder. In some embodiments, the proliferative disease is cancer. Insome embodiments the cancer is characterized by a solid tumor.

As used herein, “an effective amount” refers to the amount of eachactive agent (e.g., an immune checkpoint modulator such as an anti-PD1antibody or a fermented composition) required to confer therapeuticeffect on the subject, either alone or in combination with one or moreother active agents. In some embodiments, the therapeutic effect issuppressing cancer cell growth and/or reducing tumor burden. In someembodiments, the amount of the fermented composition is effective inenhancing the anti-cancer effect of the immune checkpoint modulator. Inother embodiments, the amount of the fermented composition is effectivein enhancing immunity of the subject against cancer cells. In someembodiments, the therapeutic effect is the prevention or inhibition oftumor growth. In some embodiments, the therapeutic effect is a decreasein a side effect associated with one or more agents/drugs. For example,a side effect that may result from inhibiting the PD-1 pathway (e.g.,fatigue, peripheral oedema, chills, pyrexia, diarrhoea, nausea,abdominal pain, cough, dyspnoea, rash, pruritus, vitiligo, arthralgia,myalgia, back pain, headache, dizziness, and/or increased aspartateaminotransferase (AST)) may be reduced by co-treatment with an modulatorof another agent (e.g., an immune checkpoint modulator and a fermentedcomposition as described herein).

Determination of whether an amount of the modulator combination achievedthe therapeutic effect would be evident to one of skill in the art.Effective amounts vary, as recognized by those skilled in the art,depending on the particular condition being treated, the severity of thecondition, the individual patient parameters including age, physicalcondition, size, gender and weight, the duration of the treatment, thenature of concurrent therapy (if any), the specific route ofadministration and like factors within the knowledge and expertise ofthe health practitioner. These factors are well known to those ofordinary skill in the art and can be addressed with no more than routineexperimentation. It is generally preferred that a maximum dose of theindividual components or combinations thereof be used, that is, thehighest safe dose according to sound medical judgment.

Empirical considerations, such as the half-life, generally willcontribute to the determination of the dosage. For example, antibodiesthat are compatible with the human immune system, such as humanizedantibodies or fully human antibodies, may be used to prolong half-lifeof the antibody and to prevent the antibody being attacked by the host'simmune system. Frequency of administration may be determined andadjusted over the course of therapy, and is generally, but notnecessarily, based on treatment and/or suppression and/or ameliorationand/or delay of a target disease/disorder. Alternatively, sustainedcontinuous release formulations of an antibody may be appropriate.Various formulations and devices for achieving sustained release areknown in the art.

In one example, dosages for an immune checkpoint modulator such as anantibody as described herein may be determined empirically inindividuals who have been given one or more administration(s) of themodulator. Individuals are given incremental dosages of the modulator.To assess efficacy of the antagonist, an indicator of thedisease/disorder can be followed.

Generally, for administration of any of the antibodies described herein,an initial candidate dosage can be about 2 mg/kg. For the purpose of thepresent disclosure, a typical daily dosage might range from about any of0.1 μg/kg to 3 μg/kg to 30 μg/kg to 300 μg/kg to 3 mg/kg, to 30 mg/kg to100 mg/kg or more, depending on the factors mentioned above. Forrepeated administrations over several days or longer, depending on thecondition, the treatment is sustained until a desired suppression ofsymptoms occurs or until sufficient therapeutic levels are achieved toalleviate a target disease or disorder, or a symptom thereof. Anexemplary dosing regimen comprises administering an initial dose ofabout 2 mg/kg, followed by a weekly maintenance dose of about 1 mg/kg ofthe antibody, or followed by a maintenance dose of about 1 mg/kg everyother week. However, other dosage regimens may be useful, depending onthe pattern of pharmacokinetic decay that the practitioner wishes toachieve. For example, dosing from one-four times a week is contemplated.In some embodiments, dosing ranging from about 3 μg/mg to about 2 mg/kg(such as about 3 μg/mg, about 10 μg/mg, about 30 μg/mg, about 100 μg/mg,about 300 μg/mg, about 1 mg/kg, and about 2 mg/kg) may be used. In someembodiments, dosing frequency is once every week, every 2 weeks, every 4weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, every9 weeks, or every 10 weeks; or once every month, every 2 months, orevery 3 months, or longer. The progress of this therapy is easilymonitored by conventional techniques and assays. The dosing regimen(including the antibody used) can vary over time.

In some embodiments, for an adult patient of normal weight, dosesranging from about 0.3 to 5.00 mg/kg may be administered. The particulardosage regimen, i.e., dose, timing and repetition, will depend on theparticular individual and that individual's medical history, as well asthe properties of the individual agents (such as the half-life of theagent, and other considerations well known in the art).

For the purpose of the present disclosure, the appropriate dosage of anantibody as described herein will depend on the specific antibody,antibodies, and/or non-antibody peptide (or compositions thereof)employed, the type and severity of the disease/disorder, whether theantibody is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantagonist, and the discretion of the attending physician. Typically theclinician will administer an antibody, until a dosage is reached thatachieves the desired result. In some embodiments, the desired result isa decrease in thrombosis. Methods of determining whether a dosageresulted in the desired result would be evident to one of skill in theart. Administration of an immune checkpoint modulator and/or thefermented composition can be continuous or intermittent, depending, forexample, upon the recipient's physiological condition, whether thepurpose of the administration is therapeutic or prophylactic, and otherfactors known to skilled practitioners. The administration of anantibody may be essentially continuous over a preselected period of timeor may be in a series of spaced dose, e.g., either before, during, orafter developing a target disease or disorder.

As used herein, the term “treating” refers to the application oradministration of a composition including one or more active agents to asubject, who has a target disease or disorder, a symptom of thedisease/disorder, or a predisposition toward the disease/disorder, withthe purpose to cure, heal, alleviate, relieve, alter, remedy,ameliorate, improve, or affect the disorder, the symptom of the disease,or the predisposition toward the disease or disorder.

Alleviating a target disease/disorder includes delaying the developmentor progression of the disease, or reducing disease severity. Alleviatingthe disease does not necessarily require curative results. As usedtherein, “delaying” the development of a target disease or disordermeans to defer, hinder, slow, retard, stabilize, and/or postponeprogression of the disease. This delay can be of varying lengths oftime, depending on the history of the disease and/or individuals beingtreated. A method that “delays” or alleviates the development of adisease, or delays the onset of the disease, is a method that reducesprobability of developing one or more symptoms of the disease in a giventime frame and/or reduces extent of the symptoms in a given time frame,when compared to not using the method. Such comparisons are typicallybased on clinical studies, using a number of subjects sufficient to givea statistically significant result.

“Development” or “progression” of a disease means initial manifestationsand/or ensuing progression of the disease. Development of the diseasecan be detectable and assessed using standard clinical techniques aswell known in the art. However, development also refers to progressionthat may be undetectable. For purpose of this disclosure, development orprogression refers to the biological course of the symptoms.“Development” includes occurrence, recurrence, and onset. As used herein“onset” or “occurrence” of a target disease or disorder includes initialonset and/or recurrence.

In some embodiments, the combination of the immune checkpoint modulatorand the fermented composition described herein are administered to asubject in need of the treatment at an amount sufficient to inhibit theactivity of one or more target signaling pathway by at least 20% (e.g.,30%, 40%, 50%, 60%, 70%, 80%, 90% or greater) in vivo. In otherembodiments, the combination is administered in an amount effective inreducing the activity level of one or more target antigens by at least20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater).

In some embodiments, a fermented composition as described herein isgiven to a subject (e.g., a human cancer patient) who has been undergoneor is under an anti-cancer treatment that involves the use of an immunecheckpoint modulator (e.g., inhibitor) as described herein.

Conventional methods, known to those of ordinary skill in the art ofmedicine, can be used to administer the pharmaceutical composition tothe subject, depending upon the type of disease to be treated or thesite of the disease. This composition can also be administered via otherconventional routes, e.g., administered orally, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir. The term “parenteral” as used hereinincludes subcutaneous, intracutaneous, intravenous, intramuscular,intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal,intralesional, and intracranial injection or infusion techniques. Inaddition, it can be administered to the subject via injectable depotroutes of administration such as using 1-, 3-, or 6-month depotinjectable or biodegradable materials and methods. In some examples, thepharmaceutical compositions are administered intraocularlly orintravitreally.

Injectable compositions may contain various carriers such as vegetableoils, dimethylactamide, dimethyformamide, ethyl lactate, ethylcarbonate, isopropyl myristate, ethanol, and polyols (glycerol,propylene glycol, liquid polyethylene glycol, and the like). Forintravenous injection, water soluble antibodies can be administered bythe drip method, whereby a pharmaceutical formulation containing theantibody and a physiologically acceptable excipients is infused.Physiologically acceptable excipients may include, for example, 5%dextrose, 0.9% saline, Ringer's solution or other suitable excipients.Intramuscular preparations, e.g., a sterile formulation of a suitablesoluble salt form of the antibody, can be dissolved and administered ina pharmaceutical excipient such as Water-for-Injection, 0.9% saline, or5% glucose solution.

In one embodiment, an antibody is administered via site-specific ortargeted local delivery techniques. Examples of site-specific ortargeted local delivery techniques include various implantable depotsources of the antibody or local delivery catheters, such as infusioncatheters, an indwelling catheter, or a needle catheter, syntheticgrafts, adventitial wraps, shunts and stents or other implantabledevices, site specific carriers, direct injection, or directapplication. See, e.g., PCT Publication No. WO 00/53211 and U.S. Pat.No. 5,981,568.

Targeted delivery of therapeutic compositions containing an antisensepolynucleotide, expression vector, or subgenomic polynucleotides canalso be used. Receptor-mediated DNA delivery techniques are describedin, for example, Findeis et al., Trends Biotechnol. (1993) 11:202; Chiouet al., Gene Therapeutics: Methods And Applications Of Direct GeneTransfer (J. A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988)263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc.Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991)266:338.

Therapeutic compositions containing a polynucleotide (e.g., thoseencoding the antibodies described herein) are administered in a range ofabout 100 ng to about 200 mg of DNA for local administration in a genetherapy protocol. In some embodiments, concentration ranges of about 500ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg,and about 20 μg to about 100 μg of DNA or more can also be used during agene therapy protocol.

The therapeutic polynucleotides and polypeptides described herein can bedelivered using gene delivery vehicles. The gene delivery vehicle can beof viral or non-viral origin (see generally, Jolly, Cancer Gene Therapy(1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, HumanGene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148).Expression of such coding sequences can be induced using endogenousmammalian or heterologous promoters and/or enhancers. Expression of thecoding sequence can be either constitutive or regulated.

Viral-based vectors for delivery of a desired polynucleotide andexpression in a desired cell are well known in the art. Exemplaryviral-based vehicles include, but are not limited to, recombinantretroviruses (see, e.g., PCT Publication Nos. WO 90/07936; WO 94/03622;WO 93/25698; WO 93/25234; WO 93/11230; WO 93/10218; WO 91/02805; U.S.Pat. Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EPPatent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virusvectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross Rivervirus (ATCC VR-373; ATCC VR-1246) and Venezuelan equine encephalitisvirus (ATCC VR-923; ATCC VR-1250; ATCC VR 1249; ATCC VR-532)), andadeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO95/00655). Administration of DNA linked to killed adenovirus asdescribed in Curiel, Hum. Gene Ther. (1992) 3:147 can also be employed.

Non-viral delivery vehicles and methods can also be employed, including,but not limited to, polycationic condensed DNA linked or unlinked tokilled adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992)3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989)264:16985); eukaryotic cell delivery vehicles cells (see, e.g., U.S.Pat. No. 5,814,482; PCT Publication Nos. WO 95/07994; WO 96/17072; WO95/30763; and WO 97/42338) and nucleic charge neutralization or fusionwith cell membranes. Naked DNA can also be employed. Exemplary naked DNAintroduction methods are described in PCT Publication No. WO 90/11092and U.S. Pat. No. 5,580,859. Liposomes that can act as gene deliveryvehicles are described in U.S. Pat. No. 5,422,120; PCT Publication Nos.WO 95/13796; WO 94/23697; WO 91/14445; and EP Patent No. 0524968.Additional approaches are described in Philip, Mol. Cell. Biol. (1994)14:2411, and in Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

Treatment efficacy for a target disease/disorder can be assessed bymethods well-known in the art.

General Techniques

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature, such as, Molecular Cloning: ALaboratory Manual, second edition (Sambrook, et al., 1989) Cold SpringHarbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methodsin Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook(J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I.Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P.Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture:Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell,eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press,Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C.Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J. M.Miller and M. P. Calos, eds., 1987); Current Protocols in MolecularBiology (F. M. Ausubel, et al., eds., 1987); PCR: The Polymerase ChainReaction, (Mullis, et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers,1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D.Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practicalapproach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000);Using antibodies: a laboratory manual (E. Harlow and D. Lane (ColdSpring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995).

Without further elaboration, it is believed that one skilled in the artcan, based on the above description, utilize the present invention toits fullest extent. The specific embodiments provided herein are,therefore, to be construed as merely illustrative, and not limitative ofthe remainder of the disclosure in any way whatsoever. All publicationscited herein are incorporated by reference for the purposes or subjectmatter referenced herein.

Example 1: Treating Colon Cancer with an Anti-PD1 Antibody andComposition X Via Oral Administration

Composition X is a fermented soybean composition prepared as follows. Anaqueous extract of soybean was prepared by conventional methods. Amixture of Lactobacillus and yeast were cultured in a medium containingthe aqueous soybean extract under conditions that allow for fermentationof the soybean extract by the microorganisms. The fermented liquid werecollected, filtered to remove solid materials, and sterilized. Theliquid solution thus prepared was concentrated to produce Composition X(in liquid form). Each milligram of Composition X contains the fermentedbroth of about 2.7 g soybean.

The effectiveness of the combined therapy of Composition X and anti-PD1antibody on colon cancer was investigated in a colon cancer mouse model.Briefly, Balb/c mice were injected subcutaneously with 2×10⁵ coloncancer CT26 cells on Day 0. The mice were injected intraperitoneallywith an anti-PD1 antibody at Day 6 at 10 mg/kg when the tumors derivedfrom the grafted colon cancer cells grew to a volume of about 60-70 mm³.The injection of the anti-PD1 antibody was repeated at the same dose atDays 8, 10, and 12. The mice were sacrificed at day 17 for analysis.During Day 0 to Day 15, some of the mice were fed with dilutedComposition X (1%, 5%, or 15%) daily and others were fed with a vehiclecontrol. A schematic illustration of this exemplary experimental designis provided in FIG. 1A.

Alternatively, Balb/c mice were fed with Composition X on a daily basisfor two weeks (14 days; starting on Day −14). The mice were theninjected subcutaneously with 2×10⁵ colon cancer CT26 cells (on Day 0).Five days after the cancer cell transplantation (Day 5), the mice weretreated with 10 mg/kg anti-PD1 via intraperitoneal injection, which isrepeated on Days 7, 9, and 11. The mice were sacrificed on Day 15 foranalysis.

As shown in Tables 1 and 2 below and FIGS. 2 and 3, Composition Xsignificantly enhanced the efficacy of the anti-PD1 antibody ininhibiting tumor growth in a dose-dependent manner as demonstrated inthe colon cancer mouse model used in this example. Table 1, FIG. 2A, andFIG. 3A show results obtained from a study following the experimentaldesign depicted in FIG. 1A. Table 2, FIG. 2B, and FIG. 3B show resultsobtained from a study following the experimental design depicted in FIG.1B.

TABLE 1 Effect of Composition X and anti-PD1 Antibody on Tumor Growth inColon Cancer Mouse Model Average Tumor Tumor Growth Group (n = 6) Volume(mm³) Inhibition (%) Vehicle 570.4 — Anti-PD1 485.8 14.8 1% CompositionX + 344.7 39.6 anti-PD1 5% Composition X + 304.8 46.6 anti-PD1 15%Composition 256.5 55.0 X + anti-PD1

TABLE 2 Effect of Composition X and anti-PD1 Antibody on Tumor Growth inColon Cancer Mouse Model Average Tumor Tumorinhibition Group Volume(mm³) rate (%) Vehicle 502.8 — anti-PD1 mAb 308.6 38.6 1% CompositionX + 182.2 63.8 anti-PD1 mAb 5% Composition X + 181.4 63.9 anti-PD1 mAb15% Composition X + 213.3 57.6 anti-PD1 mAb

On Day 15, the mice were sacrificed and splenocytes from each mouse werestained with fluorescence-labeled CD4, CD8, CD62L and CD44 antibodiesfor flow cytometry analysis. Three-color flow cytometry was performed toidentify naïve T cells (defined as CD62L+CD44low) and effector memory Tcells (defined as CD62L-CD44high).

As shown in FIG. 4, the percentages of effector memory T cells in bothsplenic CD8+ and CD4+ cells were significantly increased as a result ofthe combined treatment of anti-PD1 and Composition X, while naïve Tcells decreased as compared with the control.

Example 2: Treating Lung Cancer with an Anti-PD1 Antibody andComposition X Via Oral Administration

Composition X was prepared as described in Example 1 above. Theeffectiveness of the combined therapy of Composition X and anti-PD1antibody on lung cancer was investigated in a lung cancer mouse model asfollows.

C57BL/6 mice were injected with 5×10⁶ lung cancer LL2 cells on Day 0.Starting from Day 4, some of the mice were fed with diluted CompositionX (7.5%, 15% or 22.5% composition) once every day, and the others weretreated with a vehicle control. Starting at Day 4, when the tumor grewto a volume around 60 mm³, the mice were injected intraperitoneally with10 mg/kg an anti-PD1 monoclonal antibody once every other day (on Days4, 6, 8, 10, and 12). The Mice were sacrificed on Day 19 for analysis. Aschematic illustration of this exemplary experimental design is providedin FIG. 5.

As shown in Table 3 below and FIGS. 6 and 7, Composition X significantlyenhances the efficacy of anti-PD1 in inhibiting tumor growth in adose-dependent manner as observed in this lung cancer mouse model.Composition X acted synergistically with the anti-PD1 antibody insuppressing tumor growth.

TABLE 3 Effect of Composition X and anti-PD1 Antibody on Tumor Growth inLung Cancer Mouse Model Average Tumor Tumor Growth Group (n = 7 or 8)Volume (mm³) Inhibition (%) Vehicle 1551.9 — Anti-PD1 1354.4 12.7 15%Composition X 1179.5 24.0 7.5% Composition X + 921.0 40.7 anti-PD1 15%Composition X + 755.3 51.3 anti-PD1

Both CD4+ and CD8+ T cells infiltrated into tumor tissues were analyzedby flow cytometry as described herein. It was observed that, in bothtumor-infiltrating CD4+ and CD8+ T cells, the percentages of effectormemory T cells significantly increased as a result of the combinedtreatment, while naïve T cells decreased as compared with the vehiclecontrol. FIG. 8.

The splenic T cell profiling of the mice treated with either the vehiclecontrol or the combinations of anti-PD1 and Composition X was analyzedfollowing the methods described in Example 1 above. As shown in FIG. 9,the percentages of effector memory T cells in both splenic CD8+ and CD4+cells were significantly increased as a result of the combined treatmentof anti-PD1 and Composition X, while naïve T cells decreased as comparedwith the control.

Further, the PD1/PD-L1 expression profiling in tumor tissues wereanalyzed. Tumor-infiltrating PD-1^(high) CD4+ T cells in mice treatedwith the anti-PD-1 antibody, either taken alone or in combination withComposition X showed similar decreasing trends. Composition X alonedecreased tumor-infiltrating PD-1^(high) CD8+ T cells, which is similar(or even better than) the anti-PD-1 antibody. PD-L1 expression in tumorcells was reduced in combination therapy groups as well as (or evenbetter than) in anti-PD-1 group. FIG. 10.

MDSCs (myeloid-derived suppressor cells) are characterized asGr-1(+)CD11b(+)F4/80(+) cells. These cells play important roles in tumordevelopment and have a negative effect on tumor immunotherapy. Thecombined treatment of Composition X and anti-PD-1 significantlydecreased the percentage of MDSCs, indicating that this combined therapywould be effective in cancer treatment. FIG. 11.

Example 3: Treating Colon Cancer with an Anti-PD1 Antibody andComposition X Via Intravenous Injection

Composition X was prepared following the method described in Example 1above. The effectiveness of the combined therapy of Composition X,administrated intravenously, and anti-PD1 antibody on colon cancer wasinvestigated in a colon cancer mouse model as follows.

Balb/c mice were injected subcutaneously with 2×10⁵ colon cancer CT26cells on Day 0. The mice were injected intraperitoneally with ananti-PD1 antibody at Day 5 at 10 mg/kg when the tumors derived from thegrafted colon cancer cells reached an average size of 50 mm³. Theinjection of the anti-PD1 antibody was repeated at the same dose at Day7, 9, 11, and 13. The mice were sacrificed at day 16 for analysis. Someof the mice were dosed with 5% diluted Composition X intravenously onday 4, 7, 9, 11, 13, and 15 while some not. A group of mice wereadministrated with 15% Composition X (10 ml/kg, daily) orally from day 3until the end of the experiment with or without anti-PD1 injection forcomparison. A schematic illustration of this exemplary experimentaldesign is provided in FIG. 12.

As shown in Table 4 below and FIGS. 13 and 14, intravenousadministration of Composition X significantly enhances the efficacy ofthe anti-PD1 antibody in inhibiting tumor growth to a similar extant asoral administration of Composition X. Again, Composition X actedsynergistically with the anti-PD1 antibody in suppressing tumor growth.

TABLE 4 Effect of Intravenous Composition X and anti-PD1 Antibody onTumor Growth in Colon Cancer Mouse Model Average Tumor Tumor GrowthGroup (n = 6) Volume (mm³) Inhibition (%) Vehicle 609.5 — Anti-PD1 480.521.1 5% Composition X 415.5 31.8 (i.v.) 5% Composition X 282.4 53.7(i.v.) + anti-PD1 15% Composition X 542.3 11.0 (p.o.) 15% Composition X399.9 34.4 (p.o.) + anti-PD1

Immune cell profiling and cytokine expression in the spleen and tumorsite were analyzed by flow cytometry (FIGS. 15-22). Again, both thepercentage of effector/memory CD4+ and CD8+ T cells significantlyincreased as a result of combined treatment either by oral or byintravenous administration of Composition X at the tumor sites (FIG.15), as well as in the spleens (FIG. 16). Further, in both combinationsthe percentages of PD1^(high) CD4 and CD8 T cells were lower than thecontrol and so was the percentage of PD-L1⁺ tumor cells (FIG. 17).Similarly, the percentage of NK cells (characterized as CD3e⁻ CD49b⁺) inthe tumors were significantly higher (FIG. 18) while MDSC (characterizedas Gr-1⁺ CD11b⁺ F4/80⁺) were significantly lower (FIG. 19) in both oraland intravenous administration of Composition X combined with anti-PD1.

In addition, cytokine expression profile at the tumor site (FIG. 20-21)as well as in the serum (FIG. 22) was measured. It was observed thatboth cytokines capable of enhancing T and NK cell functions (IL-2,IFN-γ, IL-15, IL-4) and pro-inflammatory cytokines (IL-5, IL-6, TNF-α)were significantly increased both at the tumor sites and in the serum.

Example 4: Treating Colon Cancer with an Anti-PD1 Antibody andComposition X Changed the Microbiota in the Large Intestine

An anti-PD1 antibody and Composition X were given to Balb/c micetransplanted with colon cancer CT26 cells following the procedureillustrated in FIG. 1A. After the treatment, the proximal colon tissuesof the treated mice were excised on day 17, wherein the mice weresacrificed and subjected to microbiota analysis with the next generationsequencing technology. The results showed that several of the bacterialspecies were changed when mice bearing colon cancer were treated withthe anti-PD1/Composition X combination as compared to mice bearing coloncancer without any treatment. Exemplary bacterial species were listed inTable 5.

TABLE 5 Effect of Composition X and anti-PD1 Antibody on ChangingMicrobiota in Mice Bearing Colon Cancer. Average Average Proportion inanti- Kruskal- Proportion in PD1 + 15% Composition X Wallis test InputTaxon Vehicle Group Group (p < 0.05) Streptococcus 1.94E−05 0 0.0277Anaeroplasma 1.01E−05 0 0.0277 RF39 1.15E−04 0 0.0277 Ruminococcus1.78E−04 4.68E−05 0.0339

In addition, the abundance of Ruminococcus is closely related to thesize of the tumor, as shown in FIG. 23, where X-axis represents theabundance of Ruminococcus and Y-axis represent the size of the tumor.When the tumor size is bigger, i.e. the tumors in vehicle group, Theabundance of Ruminococcus in the large intestines of mice having tumorsof large size (the mice of the vehicle control group) is much higherthan that of mice having tumors of small size (the mice treated by theComposition X/anti-PD1 antibody combination). Since intestinalmicrobiota was suggested to affect the colon cancer (see, e.g., Gao et.al., Frontiers in Microbiology, 6:20, 2015), results obtained from thisstudy indicates that Composition X and/or the anti-PD1 antibody may beeffective in treating colon cancer via, inter alia, modulatingintestinal microbiota of a subject being treated with this combination.

OTHER EMBODIMENTS

All of the features disclosed in this specification may be combined inany combination. Each feature disclosed in this specification may bereplaced by an alternative feature serving the same, equivalent, orsimilar purpose. Thus, unless expressly stated otherwise, each featuredisclosed is only an example of a generic series of equivalent orsimilar features.

From the above description, one skilled in the art can easily ascertainthe essential characteristics of the present invention, and withoutdeparting from the spirit and scope thereof, can make various changesand modifications of the invention to adapt it to various usages andconditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

What is claimed is:
 1. A kit comprising: (i) an immune checkpointmodulator, which is an antibody specific to PD-1 or a ligand thereof;and (ii) a fermented soybean composition, which comprises multiplemetabolites that are generated via fermentation of soybeans by a yeast,a lactobacillus, or a combination thereof.
 2. The kit of claim 1,wherein the immune checkpoint inhibitor is an antibody specific to PD-1.3. The kit of claim 1, wherein the multiple metabolites comprise acombination of lactic acid, acetic acid, and/or 3-aminoisobutyric acid.4. The kit of claim 3, wherein the fermented composition compriseslactic acid at 5-20% by weight, acetic acid at less than 5% by weight,and 3-aminoisobutyric acid at less than 5% by weight.
 5. The kit ofclaim 1, wherein the fermented soybean composition is prepared by aprocess comprising: (i) growing a yeast, a lactobacillus, or acombination thereof in a medium comprising soybean, a portion thereof,or an extract thereof under conditions allowing for fermentation of thesoybean, the portion thereof, or the extract thereof; and (ii)collecting the fermented composition obtained from step (i).
 6. The kitof claim 5, wherein the preparation process further comprises filteringthe fermented composition obtained from step (ii), sterilizing thefermented composition, and/or concentrating the fermented composition.